The invention relates to a method for detecting mutations in samples of tissue and stool by PCR amplification using specific oligoprimers and a kit to perform the method.
An increasing body of evidence implicates somatic mutations as causally important in the induction of human cancers. These somatic mutations may accumulate in the genomes of previously normal cells, some of which may then demonstrate the phenotypes associated with malignant growth. Such oncogenic mutations may include a number of different types of alterations in DNA structure, including deletions, translocations and single nucleotide alterations. The latter, also known as point mutations, may frequently intervene in carcinogenesis, in that a variety of mutagenic chemicals induce such mutations. In addition, such mutations may occur spontaneously as a result of mistakes in DNA replication.
Advances in recombinant DNA technology have led to the discovery of normal cellular genes (proto-oncogenes and tumor suppressor genes) which control growth, development, and differentiation. Under certain circumstances, regulation of these genes is altered and they cause normal cells to assume neoplastic growth behavior. There are over 100 known protooncogenes and suppressors genes to date, which fall into various categories depending on their functional characteristics. These include, (1) growth factors and growth factors receptors, (2) messengers of intracellular signal transduction pathways, for example, between the cytoplasm and the nucleus, and (3) regulatory proteins influencing gene expression and DNA replication.
Point mutations have been directly implicated in the causation of many human tumors. Some tumors carry oncogenes of the ras gene family, which differ from their normal cellular counterpart protooncogenes by the presence of a point mutation at one of a limited number of sites in these genes. Similarly, point mutations in critical regions of tumor suppressor genes, such as p53, are often detected in tumor cells. These mutations represent qualitative changes in the tumor cell genome which distinguish these cells from normal cells and provide a basis for diagnosis of the genetic origin of a tumor under study. Identification of the mutations that have created active oncogenes may provide important diagnostic and prognostic clues for tumor development. For example, a number of mutations have been found to alter the 12th codon of the ras oncogenes, causing replacement of a normally present glycine by any of a number of alternative amino acid residues. Such amino acid substitutions create a potent transforming allele. Thus, the presence of a particular nucleotide substitution may be a strong determinant of the behavior of the tumor cell (e.g., its rate of growth, invasiveness, etc.). As a result, DNA probes of oncogene mutations have promise as diagnostic reagents in clinical oncology.
Among the various types of neoplasms, a number of those which are found in the gastrointestinal tract are associated with oncogenic mutations. This association is particularly significant for pancreatic and colorectal cancer. Colorectal cancer is the third most common malignancy in the world, with more than 700,000 new cases expected in 1996. In the United States alone, over 70,000 people will die from colorectal cancer in this same year. While patients with advanced disease have a very poor prognosis, colorectal tumors diagnosed at any stage prior to metastatis can usually be cured by surgical or colonoscopic excision. A method to detect surgically resectable tumors could, therefore, considerably reduce deaths from this disease (Winawer, et al., J. National Cancer Institute, 83:243, 1991).
A method to detect mammalian nucleic acid in stool is known from WO93/20235 (Vogelstein, B. and Kinzler, K.). This method comprises purification of faeces and two different methods for amplification of mutated ras genes. Method 1 is based upon PCR amplification of Ki-ras and subsequent cloning of the amplified Kir-ras into a bacterial phage, culturing the phage and plaque hybridizing with oligonucleotide probes which are specific for the single mutations in Ki-ras. Method 2 is based upon PCR amplification, followed by gel electrophoresis of the PCR product, hybridization of the product to the nylon filter and allele specific hybridization with isotope labeled probes, specific for each mutation. Both methods are sensitive and specific. Unfortunately these methods are both time consuming and labour intensive, since all mutations in Ki-ras are not detected by one method. More than 10 mutations of Ki-ras in exon I are already described. The above methods use gene probes specific for each of these mutations. If a stool sample shall be screened and a pirori it is not known which mutation is present, a great number of different probes has to be used in each sample, and in addition each probe has to be isotopically labeled.
PCR amplification of DNA from stool is also known from Smith-Ravin et al. (Gut, 35, 81-86, (1995)). In this procedure 10-50 g stool is used and DNA was extracted, then purified DNA was amplified using mitochondrial primers and analysed for ras mutations using an allele specific mismatch method. Total detection rate was 9 of 22, the method used great amounts of stool and was not suitable for screening since it was necessary to use specific mutant allele specific amplification (MASA) probes to detect the mutations.
In an article in Oncogene, 10, 1441-45, (1995) Hasegawa et al. have amplified DNA from 15 patients out of 19 and by using standard MASA technique, identified 3 mutations. Following this a special MASA-technique was performed, which resulted in detected mutations in 10 patients. The problem with this procedure is a low detection rate and that a specific probe has to be used for each mutation. Thus the method is not suitable for screening analysis.
Suzuki, Y. et al. (Oncogene, 5 (7): 1037-43, 1990 describes detection of ras gene mutations in human lung cancers by single stranded conformation polymorphism (SSCP) analysis of polymerase chain reaction (PCR) products. This method did, however not indicate the sequences of the mutations.
The objects of the present invention is to produce a screening procedure for detection of Ki-ras mutations in tissues and stool, with adequate specificity and simplicity to perform, using small amounts of substrate.
These objects are obtained by the present invention characterized by the enclosed claims.
The present invention is related to a screening procedure for detection of Ki-ras mutations in small samples of tissue or stool in which specific primers according to the invention is used to generate a PCR product of 221 base pairs. This product shows unique single stranded conformation polymorphism (SSCP) patterns in which each mutation is detected and identified using SSCP analyses performed on 20% homogenous polyacrylamide gels (polyacrylimide Phast(trademark) Gel, Pharmacia).
To increase the amount of the PCR product it is possible to perform subsequently semi-nested PCR in which an extra oligonucleotide was used in the 5xe2x80x2 end while the specific oligoprimer of the first step was used in the 3xe2x80x2 end. In the next PCR cyclus both oligoprimers from the first step was used which resulted in the 221 base pair PCR product In this product the mutations were detected as above with Phast gel SSCP.
Still another increase of the sensitivity according to the invention is obtained by in a third step using allele specific amplification or mutation specific primer extension. This step involves using probes containing a mismatch against the wild type gene on the last base in the 3xe2x80x2 end and an additional mismatch on base no. 2 or 3 from the 3xe2x80x2 end. This strategy surprisingly increased the sensitivity of the method. Thus by using the method according to the invention Ki-ras mutations were detected in 8 patients of 12 using double PCR and in another study 6 patients with mutations in Ki-ras of 7 patients were detected by the first step while the 7 with mutations was detected by combining step 1 and 2. Thus the detection rate was 90% with single PCR and 100% with combined PCR.
In the following the invention is disclosed in detail together with examples and figures.